Method for repairing living tissue with a hollow fiber scaffold

ABSTRACT

A scaffold of hollow fibers comprising a mixture of polylactic acid (PLA) and polybutylene succinate (PBS) and cellulose nanofibers (CNF), medical products made of these scaffolds and methods of using the scaffolds in regenerative medicine. A method for producing the scaffolds is also disclosed.

STATEMENT REGARDING PRIOR DISCLOSURES BY THE INVENTOR OR A JOINTINVENTOR

Aspects of this technology are described by Abudula, T., Saeed, U.,Memic, A. et al. in Electrospun cellulose Nano fibril reinforced PLA/PBScomposite scaffold for vascular tissue engineering. J Polym Res26, 110(Apr. 15, 2019) doi:10.1007/510965-019-1772-y which is incorporatedherein by reference in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The invention pertains to the fields of tissue engineering,electrospinning, and biocompatible scaffolding.

Description of Related Art

The design and production of scaffolds for tissue engineering byelectrospinning is a topic of great interest. This technology canproduce scaffolds composed of micro and nano fibers from a wide varietyof synthetic and natural polymers which can mimic many properties ofmaterials found in human cells; Kishan, A. P., et al., Recentadvancements in electrospinning design for tissue engineeringapplications: A review. Journal of Biomedical Materials Research Part A,2017. 105(10): p. 2892-2905.

Electrospinning is a versatile technique which can produce materialswhich can mimic native extracellular matrix (ECM), such as high surfacearea-volume ratio, and provides a substantially interconnected porousstructure; Boateng, J. S., et al., Wound healing dressings and drugdelivery systems: a review. Journal of pharmaceutical sciences, 2008.97(8): p. 2892-2923; Greiner, A, et al., Electrospinning: a fascinatingmethod for the preparation of ultrathin fibers. Angewandte ChemieInternational Edition, 2007. 46(30): p. 5670-5703. Electrospun scaffoldscan be designed to closely emulate both the tensile strength and elasticmodulus of human tissues; Grasl, C., et al., Electrospun polyurethanevascular grafts: in vitro mechanical behavior and endothelial adhesionmolecule expression. Journal of Biomedical Materials Research Part A: AnOfficial Journal of the Society for Biomaterials, The Japanese Societyfor Biomaterials, and The Australian Society for Biomaterials and theKorean Society for Biomaterials, 2010. 93(2): p. 716-723; and Pan, Y.,et al., Small-diameter hybrid vascular grafts composed ofpolycaprolactone and polydioxanone fibers. Scientific Reports, 2017.7(1): p. 3615.

Advantageously, many antimicrobial agents, growth factors, structuralmaterials such as proteins or carbohydrates, or anesthetic materials canbe easily loaded on or into electrospun materials to fully functionalizethe scaffolds for use in surgical treatments; Zhu, T., et al., Synthesisof RGD-peptide modified poly(ester-urethane) urea electrospun nanofibersas a potential application for vascular tissue engineering. ChemicalEngineering Journal, 2017. 315: p. 177-190; and Wan, X., et al.,Electrospun PCL/keratin/AuNPs mats with the catalytic generation ofnitric oxide for potential of vascular tissue engineering. Journal ofBiomedical Materials Research Part A, 2018, 106(12): p. 3239-3247.

Moreover, properly designed electrospun scaffolds allow adhesion,proliferation, and migration of human cells thus facilitatingregeneration of vascular tissue often with substantially recoveredfunction in an area in need of such regeneration. Such scaffolds andmethods are incorporated by reference to Khalf, A., et al., Celluloseacetate core-shell structured electrospun fiber: fabrication andcharacterization. Cellulose, 2015. 22(2): p. 1389-1400; or Du, J., etal., Potential applications of three-dimensional structure of silkfibroin/poly(ester-urethane) urea nanofibrous scaffold in heart valvetissue engineering. Applied Surface Science, 2018. 447: p. 269-278.

Electrospinning processes are flexible and permit many modificationsincluding those described by and incorporated by reference to Kishan, A.P. et al., Recent advancements in electrospinning design for tissueengineering applications: A review. Journal of Biomedical MaterialsResearch Part A, 2017. 105(10): p. 2892-2905 or by Phillip, M. et al.,Recent Applications of Coaxial and Emulsion Electrospinning Methods inthe Field of Tissue Engineering. BioResearch Open Access, 2016. 5(1): p.212-227.

Among such different modifications, coaxial electrospinning receivedmajor attention in biomedical applications such as tissue engineering,wound healing and controlled drug delivery. Such modifications andmethods are incorporated by reference to Kishan et al., id.; to Khodkar,F. et al., Preparation and properties of antibacterial, biocompatiblecore-shell fibers produced by coaxial electrospinning. Journal ofApplied Polymer Science, 2017. 134(25); Yoon, J., et al., RecentProgress in Coaxial Electrospinning: New Parameters, Various Structures,and Wide Applications. Advanced Materials, 2018. 30(42): p. 1704765; andto Nguyen, T. T. T., et al., Porous core/sheath composite nanofibersfabricated by coaxial electrospinning as a potential mat for drugrelease system. International journal of pharmaceutics, 2012. 439(1-2):p. 296-306.

Despite the flexibility and other advantages of electrospinningprocesses, few studies report the development of a hollow fibrousstructure by electrospinning due to the complexity in preparing andcharacterizing hollow electrospun fiber structures; Lee, G. H., et al.,Controlled wall thickness and porosity of polymeric hollow nanofibers bycoaxial electrospinning. Macromolecular Research, 2010. 18(6): p.571-576.

This is a significant problem because hollow fibrous scaffolds arehighly desirable in many tissue engineering applications. A hollowelectrospun fiber can generate higher surface area and porosity comparedto a solid fibrous scaffold and hollow electrospun fibers can containpores that not only increase their surface area by which enhancepermeability of, and facilitate nutrient delivery through, the hollowelectrospun fibers. These properties promote and enhance uniformcellular growth and proliferation throughout and around the scaffold, aswell as facilitate removal of cellular debris and byproducts of degradedscaffolds; see Yoon, J., et al., Recent Progress in CoaxialElectrospinning: New Parameters, Various Structures, and WideApplications. Advanced Materials, 2018. 30(42): p. 1704765 and Tuin, etal., Interconnected, microporous hollow fibers for tissue engineering:Commercially relevant, industry standard scale-up manufacturing. Journalof Biomedical Materials Research Part A, 2014. 102(9): p.3311-3323.Zhang, Y., et al., Preparation of core-shell structuredPCL-r-gelatin bi-component nanofibers by coaxial electrospinning.Chemistry of Materials, 2004. 16(18): p. 3406-3409; Nagiah, N., et al.,Highly compliant vascular grafts with gelatin-sheathed coaxiallystructured nanofibers. Langmuir, 2015. 31(47): p. 12993-13002; and Tuin,S. A., et al., Interconnected, microporous hollow fibers for tissueengineering: Commercially relevant, industry standard scale-upmanufacturing. Journal of Biomedical Materials Research Part A, 2014.102(9): p. 3311-3323.

In view of the limitations of existing electrospun materials, includingthe inability to adequately diffuse nutrients and oxygen to regeneratingtissues, which often result in failure of a tissue repair to grow orregenerate, or even necrosis and cell death, the inventors sought todevelop a new multifunctional material comprising a combination ofpolylactic acid (“PLA”), polybutylene succinate (“PBS”), and cellulosenanofibers (“CNF”) that would mimic native extracellular matrix (“ECM”),serve as vascular prostheses, and/or exhibit other advantageousproperties such as acting as an artificial vascular system deliveringnutrients to regenerating tissue thus enhancing tissue regeneration.

SUMMARY OF THE INVENTION

The foregoing paragraphs have been provided by way of generalintroduction, and are not intended to limit the scope of the followingclaims. The described embodiments, together with further advantages,will be best understood by reference to the following detaileddescription taken in conjunction with the accompanying drawings.

Among its other aspects, the invention is directed to a CNF reinforced,homogenously blended, microporous hollow fibrous PLA/PBS scaffoldsuitable and to methods of using this scaffold for tissue regenerationand for vascular tissue engineering.

Another aspect of the invention is directed to a method for coaxialelectrospinning to produce these hollow fibrous composite scaffoldshaving the physical and functional properties disclosed herein.

Embodiments of the invention include, but are not limited to thefollowing.

One aspect of the invention is directed to a homogenous scaffoldcomprising, consisting essentially of, or consisting of fibers having ahollow core and a peripheral shell, wherein the peripheral shellcomprises polylactic acid (PLA), polybutylene succinate (PBS) andcellulose nanofibers (CNF). Homogenous mixing of the PLA, PBS and CNFwithin the nanofibers of the scaffold in different volumes or segmentsof the fiber different in composition by no more than 0.1, 0.2, 2.5,0.5, 1, 2, 5 or 10% determined by X-ray photoelectron spectrometry(“XPS”). A maximum weight percentage of CNF in a PLA/PBS matrix is 2.5%which defines the coherency in the shell polymer.

In an alternative embodiment, the scaffold may comprise solid fibers ofPLA and PBS containing CNF in the same amounts disclosed herein for thecorresponding hollow fibers.

In another alternative embodiment, the scaffold may comprise hollow orsolid fibers of 60 to 40 wt. % PLA and 40 to 60 wt. % PBS without CNF.Such fibers are produced by electrospinning without a core material suchas glycerol or mineral oil. In one embodiment the feed rate for a stocksolution comprising PLA and PBS into an electrospinning device may rangefrom 0.2, 0.5, 1.0, 1.5 to 2.0 mL/hr.

Typically the peripheral shell of the hollow fibers comprises 40, 45,50, 55 to 60 wt. % PLA and 60, 55, 45, or 40 wt. % of a mixture of PBSand CNF, based on a total weight of the PLA, PBS and CNF in theperipheral shell. The mixture of PBS and CNF typically contains 1, 2, 3,4 to 5 wt. % CNF based on the combined weight of the PBS and CNF. Shellstock solutions may contain higher concentrations of CNF depending onthe amount of solvent so as to provide a final CNF concentration in theperipheral shell between about 1 and 5 wt. %.

In one embodiment, peripheral shell of the hollow fibers comprise amixture of about 50 wt. % PLA and about 50 wt. % of a mixture of PBS andCNF, based on a total weight of the PLA, PBS and CNF, wherein saidmixture of PBS and CNF contains 1, 2 or 3 wt. % CNF, preferably about 2wt. % CNF based on the combined weight of the PBS and CNF.

The hollow fibers of the scaffold may have average diameters rangingfrom 400, 500, 600, 700, 800, 900 to 1,000 nm and the hollow core has adiameter ranging from 300, 400, 500, 600, 700, 800 to 900 nm. In oneembodiment the fibers have an average diameter ranging from 500, 600,700 to 800 nm and the hollow core has a diameter ranging from 400, 500,600 to 700 nm. A preferred diameter of the shell ranges from 600 to 1000nm and a preferred diameter of the hollow core diameter ranges from 200to 500 nm. The range of diameter typically depends on the feed rate ofthe shell and core solution.

The peripheral shell of the fibers of the scaffold may compriseuniformly distributed pores ranging from about 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95 to 100 nm in diameter. The hollownessof the fibers and/or their porosity permits nutrient solutions, such ascell culture medium, or other soluble agents to permeate through thefibers.

In one embodiment the diameter of the hollow fibers ranges from about580, 600, 620, 640, 660, 680, 700, 720 to about 730 nm; the core shellthickness of the fibers ranges from about 290, 300, 320, 340, 360, toabout 365 nm and the core shell comprises uniformly distributed poresabout 40, 45, 50, 55 to about 60 nm in diameter and has a wettabilitycharacterized by a water contact angle of less than about 35, 40, 45,50, 55, or 60 degrees. In a related embodiment, these hollow fibers areproduced by, and have the features derived from, electrospinning a corematerial comprising glycerol at a core feed rate of about 0.07, 0.08 or0.09 mL/hr and a shell stock solution comprising PLA, PBS and CNF at ashell feed rate of about 0.9, 1.0 to 1.1 mL/hr.

Another embodiment is directed to a scaffold comprising hollow fibershaving average diameters ranging from about 600, 650, 700 to about 750nm, a core shell thickness ranging from to about 310, 320, 340, 360, 380to about 400 mm wherein said core shell comprises uniformly distributedpores about 35, 45, 50, 55 to about 60 nm in diameter, and a watercontact angle of less than 40, 45, 50, 55 or 60 degrees. In a relatedembodiment, these hollow fibers are produced by, and have the featuresderived from, electrospinning a core material comprising mineral oil ata core feed rate of about 0.09, 0.1 to 0.11 mL/hr and a shell stocksolution comprising PLA, PBS and CNF at a shell feed rate of about 0.9,1.0 to 1.1 mL/hr.

A related embodiment is directed to a composition comprising thescaffolds as disclosed herein. Such compositions include a sterile oraseptic patch, packing, dressing or bandage comprising the scaffold ormesh as disclosed herein and, optionally, may be coated with orotherwise contain cells or one or more biologically active agents orstructural materials. In some embodiments the scaffolds may be preseededwith stem cells or other partially or fully differentiated cells,including autologous, allogenic, or xenogeneic cells or harvested nativecells or cultured cells including but not limited to cultured stemcells. Alternatively a scaffold may be exposed to regenerative cellsonce placed in situ so that cells attach or move into the scaffold. Suchcells may migrated from adjacent bodily tissues or blood or be injected.Such cells may be alive and capable of division or rendered incapable ofdivision by exposure to a chemical or radiological agent. In someembodiments such cells will embryonic stem cells, bone marrow stem cells(BMSCs), or mesenchymal stem cells (MSCs) or other cells capable ofregenerating damaged tissue.

A scaffold may also be coated or otherwise contacted with a biologicallyactive agent such as or more hemostatic agents, cytokines, growthfactors, desiccants, vitamins, antimicrobial agents, analgesics,anti-inflammatory agents, or combinations thereof. Other coatingsinclude structural materials such as proteins like fibrin or collagen orother components of the ECM.

Another aspect of the invention is directed to a method for repairing,regenerating or otherwise healing or growing a tissue comprisingapplying the scaffold as disclosed herein on, in or around a tissue inneed of repair. Any tissue may benefit for use of the scaffold asdisclosed herein, especially vascularized tissues in need of a readysupply of oxygen and other nutrients delivery of which can be enhancedvia a scaffold. In some embodiments a scaffold may be surgicallyimplanted or imposed on a target tissue or organ and in others it may beinjected or inserted laparoscopically.

In some embodiments, the scaffold may be preseeded with stem cells orcells similar or identical to the tissue or organ in need ofregeneration. In other embodiments a scaffold once in place may becomepopulated with cells that migrate into it or otherwise attach to it andhelp heal or regenerate a target tissue.

The tissue in need of repair may be a vascularized tissue containingarteries, veins, capillaries or lymphatic vessels. Highly vascularizedtissues include muscle tissue, lung tissue and liver tissue. Thescaffold as disclosed herein may be used to vascular or endovasculartissues such as those forming the arteries, veins, capillaries and hearttissues. It may be used to heal or regenerate tissues of the heart,kidney, liver, spleen, pancreas, bladder, skeletal muscle, small bowel,large bowel, stomach, bone, brain, or lung.

The scaffold may be used in reconstructive surgery or treatment of acuteor chronic wounds or in orthopedic surgeries such as those involvingspine diseases, sports injuries, degenerative diseases, infections,tumors, and congenital disorders. It may be used to treat skin or othertissue that has been wounded, punctured, lacerated, crushed, burned orotherwise damaged. It may be used to treat surgical wounds includingthose resulting from plastic surgery. It may be used during treatment ofbone fractures or correction of bone defects or other surgeriesinvolving bone. It may be used to facilitate healing or regeneration ofthe jaw or teeth.

In another embodiment, the scaffold as disclosed herein may be used totreat or enhance healing or regeneration of a poorly vascularized oravascular tissues such dense connective tissue or cartilage.

In some embodiments, the scaffold may be used to regenerate or grow atissue ex vivo or in vitro for study or for reimplantation.

Another embodiment of the invention involves a method for producing ascaffold, of hollow fibers comprising co-axially electrospinning fiberswhich contain a core and a peripheral shell; wherein the core comprisesglycerol, mineral oil or another immiscible (with the shell solution)fluid, and the shell comprises a mixture of polylactic acid (PLA),polybutylene succinate (PBS) and cellulose nanofibers (CNF); andremoving the glycerol or mineral oil from the core and volatiles fromthe shell.

In some embodiments of this method the co-axial electrospinningcomprises feeding glycerol or mineral oil (or another liquid immisciblewith a shell forming solution) to an inner layer of a co-axial spinneretat a core feed rate ranging from about 0.05, 0.06, 0.07, 0.08, 0.09 to0.1 mL/hr and feeding a shell forming solution comprising PLA, PBS andCNF into an outer layer of the spinneret at a shell feed rate rangingfrom 0.5 to 2.0 mL/hr. In some embodiments of this method the core feedrate is 0.08, 0.09, 0.10, 0.11, to 0.12 mL/hr and the shell feed rate is0.8, 0.9, 1.0, 1.1 to 1.2 mL/hr.

During electrospinning the resulting fibers are stretched. Typically,the fibers are stretched over a distance of 6, 8, 10, 12, 14, 16, to 18cm at a voltage ranging from 15, 20 to 25 kV.

In one embodiment the shell forming solution comprises shell componentsin an amount ranging 40, 45, 50, 55 to 60 wt. % PLA and 60, 55, 50, 45to 40wt. % of a mixture of PBS and CNF, based on a total weight of thePLA, PBS and CNF; wherein said mixture of PBS and CNF contains 1, 2, 3,4 to 5 wt. % CNF based on the combined weight of the PBS and CNF; andwherein said solution comprises at least one organic solvent dissolvingthe shell components. In one embodiment the shell-forming solutioncomprises about 40, 50 or 60wt. % of the shell components (PLA andPBS/CNF), preferably about 50 wt. %, and wherein the at least oneorganic solvent dissolving the shell components is a mixture ofchloroform and ethanol at a volumetric ratio of 2:1, 3:1, or 4:1preferably about 3:1.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 schematically illustrates one embodiment of hollow fiberpreparation and design based on a coaxial electrospinning method. Coresolution 100 and shell solution 200 are fed to a spinneret 300 whichcomprises a spinneret fitting 310 and a double layer capillary 320,electric force 400 draws the materials out of the spinerette andproduces a core-filled filament 510. The core is removed by evaporationstep/evaporator 500 forming a hollow fiber 520.

FIGS. 2A-2B. Cross-section by SEM of hollow fibrous scaffolds made ofglycerol at a 1 ml/h shell feed rate and at a 0.03 ml/hr core feed rate.

FIGS. 2C-2D. Cross-section by SEM of hollow fibrous scaffolds made ofglycerol at a 1 ml/h shell feed rate and at a 0.05 ml/hr core feed rate.

FIGS. 2E-2F. Cross-section by SEM of hollow fibrous scaffolds made ofglycerol at a 1 ml/h shell feed rate and at a 0.08 ml/hr core feed rate.

FIGS. 2G-2H. Cross-section by SEM of hollow fibrous scaffolds made ofglycerol at a 1 ml/h shell feed rate and at a 0.10 ml/hr core feed rate.

FIGS. 2I-2J. Cross-section by SEM of hollow fibrous scaffolds made ofglycerol at a 1 ml/h shell feed rate and at a 0.12 ml/hr core feed rate.

FIGS. 2K-2L. Cross-section by SEM of hollow fibrous scaffolds made ofglycerol at a 1 ml/h shell feed rate and at a 0.15 ml/hr core feed rate.

FIGS. 3A-3B. Cross-section by SEM of hollow fibrous scaffolds made ofmineral oil at a 1 ml/h shell feed rate and at a 0.08 ml/hr core feedrate.

FIGS. 3C-3D. Cross-section by SEM of hollow fibrous scaffolds made ofmineral oil at a 1 ml/h shell feed rate and at a 0.10 ml/hr core feedrate.

FIGS. 3E-3F. Cross-section by SEM of hollow fibrous scaffolds made ofmineral oil at a 1 ml/h shell feed rate and at a 0.12 ml/hr core feedrate.

FIGS. 3G-3H. Cross-section by SEM of hollow fibrous scaffolds made ofmineral oil at a 1 ml/h shell feed rate and at a 0.15 ml/hr core feedrate.

FIGS. 4A-4D. SEM images of hollow fibrous composite scaffolds: GC (FIG.4A-4B) and OC (FIG. 4C-4D). See Table 1 for description of GC and OCscaffolds.

FIGS. 5A-5F illustrate fiber size distributions of the solid scaffolds50/50 (FIG. 5A) and C4 (FIG. 5B); and the hollow scaffolds GH (FIG. 5C),GC (FIG. 5D), OH (FIG. 5E) and OC (FIG. 5F). See Table 1 for descriptionof 50/50, C4, GH, GC, OH, and OC scaffolds.

FIGS. 6A-6D illustrate a high resolution SEM images of hollow fibrousscaffolds: GH (FIG. 6A), OH (FIG. 6B), GC (FIG. 6C), and OC (FIG. 6D).

FIGS. 7A-7D. Deconvoluted XPS spectra of hollow fibrous scaffolds: GH(FIG. 7A), OH (FIG. 7B), GC (FIG. 7C), and OC (FIG. 7D).

FIG. 8. Water contact angles of hollow fibrous scaffolds (GH, OH, GC,OC) compared to the solid fibrous scaffolds (50/50, C4).

FIG. 9. Protein adsorption capacities of hollow fibrous scaffolds (GH,OH, GC, OC) compared to the solid fibrous scaffolds (50/50, C4). TheY-axis unit is “adsorbed protein in μg/mg and shows the amount ofprotein adsorbed in each mg of scaffold.

FIGS. 10A-10D. GFP/DAPI stained images of GH (FIG. 10A), OH (FIG. 10B),GC (FIG. 10C) and OC (FIG. 10D) hollow fibrous scaffolds after 7 days ofcell culturing.

FIG. 11A. MTT optical density value for the hollow fibrous scaffoldafter one and two weeks of cell culturing.

FIG. 11B. Viability comparison after one week between solid and hollowfibrous scaffolds.

FIG. 12A. AFM surface topography of electrospun PLA/PBS single fiber.

FIG. 12B. AFM surface topography of electrospun PLA/PBS/CNF compositesingle fiber.

DETAILED DESCRIPTION OF THE INVENTION

The inventors recognized the significant disadvantages of using solidfibrous scaffolds for tissue engineering as these induce the formationof necrotic cores within a scaffold designed to induce recovery orregeneration in large tissue defects. This is because the boundary ofthe scaffold is often blocked by seeded cells, hence vital nutrientscannot be transported properly to cells in the central area of thescaffolds.

In contrast, the microporous walls and hollowness in the hollow fiberscaffolds disclosed herein provide external channels for mass transportto ensure the cellular growth throughout the scaffold. Therefore, thecell viability, growth rate and tissue recovery rate are greatlyenhanced.

Surprisingly, compared to corresponding solid fiber scaffolds, theformation of hollow fiber scaffolds showed a significant decrease in thecontact angle and wettability. In terms of contact angle andwettability, as shown herein, the morphological structure of thePLA/PBS/CNF hollow fiber scaffolds was determined to be more significantthan their composition. It was found that both PLA and PBS exhibithydrophobic surface properties, but their 50/50 wt. % combinationreduced the water contact angle by more than 30°. Surprising it was alsofound that the incorporation of CNF into the PLA/PBS fiber also reducedthe water contact angle; compare 50/50 with C4 in FIG. 8.

Furthermore, protein adsorption results in FIG. 9 showed that the largesurface area, which resulted from the use of microporous hollow fibrousscaffold, attracted more protein to the scaffold surface and that theincorporation of hydrophilic nano cellulose fibrils (CNF) furtherenhanced protein adsorption. Enhancement of protein adsorption to ascaffold can provide a microenvironment for sufficient cell-cellinteraction, cell migration, proliferation and differentiation requiredfor tissue repair or regeneration.

Surprisingly, mechanical testing results showed that strength,elasticity and hardness of the hollow fibrous scaffolds were notsignificantly different than those of the solid fibrous scaffold.

The flexibility and ductility of the hollow fibrous scaffolds was muchlower when compared to a stiffer solid fibrous scaffold. This may be dueto an increase in the number of interfaces in the hollow fibrousscaffolds which can act as crack initiation or stress concentrationsites. The incorporation of cellulose nanofibrils into hollow fibers notonly significantly increased the strength and elastic modulus of thescaffold but also improved the flexibility. In contrast for the solidfiber, ductility and flexibility of the scaffold were not significantlyaffected by the presence of cellulose nanofibrils.

Polylactic acid (“PLA”), an aliphatic polyester, is a widely studiedsynthetic polymer for use in tissue engineering due to its renewability,biodegradability and cost effectiveness; O'Brien, F. J., Biomaterials &scaffolds for tissue engineering. Materials today, 2011. 14(3): p.88-95; Gigli, M., et al., Poly (butylene succinate)-based polyesters forbiomedical applications: A review. European Polymer Journal, 2016. 75:p. 431-460; and Hardiansyah, A., et al., Electrospinning andantibacterial activity of chitosan-blended poly(lactic acid) nanofibers.Journal of Polymer Research, 2015. 22(4): p. 59m each incorporatedherein by reference in their entirety.

Polybutylene succinate (“PBS”) is an aliphatic polyester polybutylenethat is flexible, has a high degree of crystallinity, biodegradable, andhas cell-friendly surface characteristics.

The inventors have recognized that PBS, as a relatively soft polymercompared to PLA, can provide a plasticizing effect on the mechanicallystiff but brittle PLA.

As disclosed herein, it is possible to reinforce a PLA/PBS matrix usingcellulose nanofibrils (CNF) which can be synthesized from the mostabundantly found natural polymer cellulose; Benitez, A. and A. Walther,Cellulose nanofibril nanopapers and bioinspired nanocomposites: a reviewto understand the mechanical property space. Journal of MaterialsChemistry A, 2017. 5(31): p. 16003-16024; and Hanif, Z., et al.,Butanol-mediated oven-drying of nanocellulose with enhanced dehydrationrate and aqueous re-dispersion. Journal of Polymer Research, 2017.25(3): p. 191, each incorporated herein by reference in their entirety.

In some embodiments, the cellulose nanofibrils or CNF will have lengthsranging from 1, 2, 3, or >3 μm, diameters ranging from <0.5, 0.5, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or 30 nm as measured byTEM.

In some embodiments, the ratio of length to diameter of a CNF may rangefrom 200:1, 150:1 to 100:1. In some embodiments, CNF can exhibit amechanical performance characterized by an elastic modulus in the rangeof 40-50 GPa, preferably about 46.6 GPa and a tensile strength in therange of 1000 to 1200 MPa, preferably about 1170 MPA. Physicalproperties of CNF are incorporated by reference to Clemons, C.,Nanocellulose in spun continuous fibers: A review and future outlook.Journal of Renewable Materials, 2016. 4(5): p. 327-339. Typically, theCNF are ultrathin nanofibrils with large length to width aspect ratio.The average width of CNF is 50±10 nm and length is 2000±240 nm resultingin an aspect ratio of L/w=40±4. The average ratio of diameter to lengthis 0.025±0.002. During the electrospinning process single CNFs wereuniformly dispersed into PLA/PBS matrix without agglomeration. Thephenomenon can be identified from atomic force microscopy (AFM) images;see FIGS. 12A and 12B.

The nanoscale characteristics of CNF are helpful in achieving highmagnitude of orientation and high moisture adsorption capacity which arefavorable for medical applications. Moreover, it is advantageouscompared to other reinforcing agents such as carbon nanotube in terms ofbiocompatibility and bioactivity.

An AFM analysis was performed on the CNF incorporated PLA/PBS andnoticed that CNF is not only conjugated into the PLA/PBS matrix but alsoCNF orientation is exactly parallel to the electrospun fiber; see FIGS.12A and 12B. This implies that the orientation of CNF can be tailored byelectrical charge during the process of electrospinning.

The moisture adsorption capacity of CNF ranges from about 13 to 24 gwater/g CNF. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) assay test was performed on the electrospun scaffolds. TheCNF contained scaffolds supported cell attachment and growth withoutshowing any adverse effects. The cell growth rate on the CNF reinforcedcomposite was much better than for the control reference. These resultsshow that the CNF is 100% biocompatible making its suitable forbiomedical applications. In view of these advantageous properties, it isnot necessary to incorporate other types of fibers, fillers or particlesnon-CNF forms of cellulose, such as cellulose nanoballs or nanopellets,sisal or hemp fibers, or shorter cellulose nanowhiskers, cellulosenanocrystals, cellulose nanorods, which have needle-like or rod-likemorphologies and have shorter length to diameter ratios than CNF; orother organic or inorganic fillers such as silver or other metals, orclays into the fibers as disclosed herein.

Porosity. Scaffolds per se or the walls of the scaffold fibers maycontain pores, preferably uniformly distributed pores where the numberof pores in different areas of the fiber vary by no more than 5, 10, 15,or 20%. In most embodiments, the sizes of the pores in the walls of thescaffold fibers will be no more than 20, 30, 40, 50, 60, 70, 80, 90 or100 nm, preferably from about 40 to about 60 nm.

In some embodiments, the scaffolds may contain pores ranging from >100,200, 500 to 1,000 nm, or in scaffolds, at least 1, 2, 5 or 10 μm.Preferably, the pores in the scaffold are uniformly sized anddistributed, for example, where the average size or numbers of pores indifferent areas of the scaffold or scaffold fiber walls differ by nomore than 1, 2, 5, 10, or 20% in average area or average number. Apartfrom the porosity of the fiber walls, the scaffold itself has a porosityranging from about 3, 4, 5, 6, 7, 8, 9, to 10 um. The developed scaffoldis an interconnected macroporous structure having average pore size ofabout 3.5 μm.

Wettability of the scaffold or its fibers can be determined bymeasurement of the water contact angle. In some embodiments, the watercontact angle of a scaffold as disclosed herein, ranges from not morethan 10, 25, 30, 35, 40, 45, to 50° or any intermediate value withinthis range. Typically, the water contact angle of a scaffold of hollowfibers as disclosed herein will be less than that of a scaffold ofcompositionally similar solid, non-hollow fibers. As disclosed hereinthe inventors have also found that a combination of PLA and PBS (e.g.about a 40/60, 50/50 or 60/40wt. % combination) reduces the watercontact angle of the hollow fibers, for example by at least 10, 20, or30 degrees when compared to a solid fiber

A scaffold or mesh, such as a scaffold used for tissue engineering, is astructure made of artificial or natural substances that acts as a shapeon which cells can grow. A scaffold can be inert and not interact withthe cells growing on it, or it can actively help the cells to grow byreleasing chemical signals and/or nutrients. A scaffold can be partiallyor completely biodegradable or non-biodegradable. Typically a scaffoldas disclosed herein comprises hollow fibers made of PLA, PBS and CNF, iswettable, and has a surface roughness and porosity that permits it tobind to proteins and cells. The hollowness and porosity of the fiberspermits enhanced transport of nutrients to cells growing on thescaffold. These features permit it to provide a suitable biocompatiblemicroenvironment for the sufficient cell-cell interaction, cellmigration, proliferation and differentiation. In preferred embodimentsthe scaffold as disclosed herein is biodegradable.

The thickness of a flat scaffold or conformation of a shaped scaffoldmay be selected based on the type of tissue being repaired orregenerated. In some embodiments, the thickness of a scaffold asdisclosed herein will range from <0.1, 0.1, 0.2. 0.5, 1, 2, 5, 10 or >10mm. Preferably, a flat sheet scaffold has a thickness of 0.05, 0.1, 0.2,0.5, 1.0 to 2 mm which is mainly dependent on electrospinning time.

In some embodiments, the scaffold may be coated with or otherwiseincorporate one or more therapeutic, prophylactic, or diagnostic agents,such as hemostatic agents, anti-infectives, growth factors, cytokines,stem cells, cultured cells such as induced stem cells, or other cellssuch as autologous or allogenic cells, anesthetics, vasoconstrictors,ECM components such as collagen (including Types 1, 2, 3, 4 and 5),fibrin, chitosan, glycosaminoglycan including hyaluronic acid, laminin,or fibronectin, antibodies or complement factors. These additionalcomponents may be coated or attached on the surfaces of, or in thehollow cores of, electrospun fibers.

Electrospinning procedures. Electrospinning uses an electrical charge todraw very fine (typically on the micro or nanoscale) fibers from aliquid. One skilled in the art may select a commercially availableelectrospinning device equipped to feed the core and shell solutionsinto the inner and outer layers of spinnerets sized to produce thehollow fibers as disclosed herein. Core-shell fibers may be produced byusing a co-axial nozzle for electrospinning as described by andincorporated by reference to Yarin A L, et al. Evolution of core-shellstructure: From emulsions to ultrafine emulsion electrospun fibers. J.Mater. Chem. 2007; 17: 2585. The co-axial nozzle comprises two cylinderswith one cylinder situated within the core of a larger bore cylinder.Two different solutions are dispensed simultaneously through the innerand outer cylinders and charged in the same way as conventional singlebore nozzle. There are several parameters that can be modified tocontrol the size of the fibers and the volume ratio of the core andshell material. Fiber diameter can be controlled by the nozzle diameterwhile the volume ratio is by the feed-rate of the core and shellsolution as described by and incorporated by reference to Chakraborty S,et al., Electrohydrodynamics: A facile technique to fabricate drugdelivery systems. Adv Drug Deliv Rev 2009; 61: 1043. Those skilled inthe art may select an appropriate electrospinning device, for example,from those which are commercially available. Such devices are available,and incorporated by reference to, the Electrospinning Device Cataloguehypertext transferprotocol://electrospintech.com/espincatalogue.html#.Xg4B de-WzIU (lastaccessed Jan. 2, 2020).

Electrospinning stock solutions used to make the hollow fibers asdisclosed herein typically comprise, for the core feed, glycerol,mineral oil, or another solvent that is immiscible with the stocksolution for the shell. However, glycerol and mineral oil may beslightly soluble in some shell stock or precursor solutions as long asthe degree of solubility does not significantly affect electrospinningof the core and shell.

As shown herein electrospinning is a versatile technique to producefibrous scaffold which mimics the native extracellular matrix (ECM) forvascular prosthesis because conventional solid fibrous scaffolds lackthe ability of nutrient diffusion and have poor permeability resultingin the cell death and tissue necrosis. A hollow fibrous scaffold cangreatly improve permeability and nutrient diffusion throughout thescaffold. The hollow fibers have vessel-like micro or nano structureswhich can serve as an excellent artificial vascular system.

The inventors have developed scaffolds of hollow fibers comprising aunique combination of cellulose nanofibrils (CNF) reinforcing a PLA/PBScomposite which are produced by a coaxial electrospinning techniqueforming a homogenous blended and hollow microporous fibrous structure.Glycerol and mineral oil were used as core templates which were removedby evaporation. Both templates allowed hollow fiber formation anduniform pore dispersion in the walls of the hollow fibers occurred whenthe mineral oil was used. The scaffold made by mineral oil templateshowed an enhanced wettability and protein adsorption capacity which maybe due to the pore size distribution in the hollow fibers. These hollowfibrous scaffolds demonstrated superior ability to permit cellattachment and cellular proliferation as shown by a cell culture testand should provide a more favorable microenvironment for regeneration oftissues, such as to regenerate healthy vascular tissue.

EXAMPLE

Materials. Polylactic acid (PLA 2003D) was purchased from Nature Works,USA. Poly butylene succinate (PBS, commercial name is Bionolle) wasobtained from Showa Denko, Japan. Cellulose Nanofibrils (CNF) wasacquired from University of Maine, USA. Dulbecco's Modified Eagle'sMedium (DMEM) was purchased from Lonza, USA. Proteinase

K solution (20 mg/m1), Dulbecco's phosphate-buffered saline and 10%fetal bovine serum (FBS) was purchased from Thermo Fisher ScientificInc., Fair Lawn, N.J. Glycerol was obtained from Loba Chemie Pvt Ltd,India. Mineral oil (P3) is from Pfeiffer Vacuum, Germany. Chloroform,ethanol, sodium dodecyl sulfate (SDS), Lysozyme, tris buffer solution(0.1 M, pH-7), calcium chloride (CaCl₂) and sodium azide (NaN₃) werefrom Sigma-Aldrich, St. Louis, Mo., USA.

Preparation of hollow fibrous scaffold. A mixture of PLA and PBS at aweight ratio of 50/50 was dissolved in chloroform and ethanol with 3:1volume ratio by stirring for 3 hours.

In order to incorporate CNF into PLA/PBS, CNF/PBS composites with 4% ofCNF concentrations were prepared first by melt extrusion. Then, themixture of PLA and CNF/PBS composite in equal weight ratio was dissolvedin the same solvent system overnight.

A Nanon 101A electrospinning setup (NANON Supply, MECC, Fukuoka, Japan)was adapted for Coaxial Electrospinning. An ultra-thin coaxial spinneret(NANON Supply, MECC, Fukuoka, Japan) was used to create coaxialelectrospun fibers. The PLA/PBS solution or the composite solution wasdelivered to outer layer of the spinneret by a system provided with asyringe pump using a Teflon®(Polytetrafluoroethylene)tube at 1 mL/h offeed rate.

An extensional syringe pump (KDS 100, KD Scientific Inc., USA) connectedwith Teflon® tube was used to deliver the core materials into a 27-gaugeblunt metallic needle.

The double layer solution was electrically stretched at 20 kV of voltageover 12 cm of distance and finally collected on flat aluminum sheet.

Two types of materials were selected for the core layers which wereglycerol and mineral oil. Initial adjustments were performed on bothmaterials using PLA/PBS solution as a shell layer.

After electrospinning, the collected samples were placed in oven at 55°C. to evaporate the core solution. It was found that full evaporation ofglycerol can be achieved in 24 hours while 5˜6 days were needed toevaporate the mineral oil from the sample. The prepared samples werenamed according to their core-shell composition before drying, as shownin Table 1.

TABLE 1 Abbreviated name of hollow fiber, according to their core-shellcomposition Shell feed Scaffold Core Core feed rate rate Name material(mL/h) Shell material (mL/h) GH Glycerol 0.08 PLA/PBS 1.0 GC Glycerol0.08 PLA/PBS/CNF 1.0 OH Mineral oil 0.1 PLA/PBS 1.0 OC Mineral oil 0.1PLA/PBS/CNF 1.0 50/50 — — PLA/PBS 0.5 (solid) C4 — — PLA/PBS/CNF 0.5(solid)

Characterization. The morphological features of the prepared fibers wereobserved using a Field emission scanning electron microscopy (FESEM,JEOL JSM 7600F, Tokyo, Japan). A customized image processing method wasemployed to calculate size distribution of the fibers. The SEM imageswere processed through adjustment, Gaussian smoothing, localthresholding and noise removal. Additionally, the fiber sizes werecalculated by Canny Edge based Euclidian distance transform.

The cross-section of the hollow fibrous scaffold was evaluated using SEMto confirm fiber hollowness and measure the inner diameter. The scaffoldwas frozen in liquid nitrogen and cut with a razor blade. The coreremoval was performed after cutting the sample and mounting it on theSEM specimen in order to avoid any deformation of the fiber crosssection.

The XPS measurements for the prepared scaffolds were carried out in anultra-high vacuum multi-technique surface analysis system (SPECS GmbH,Germany). A standard dual anode X-ray source SPECS XR-50 with Mg-Kα,1283.6 eV was used to irradiate the sample surface. The power of X-rayon the sample surface was 100 W and a take-off-angle for electronsrelative to sample surface plane was 90°. The pressure in the analysischamber was kept at 5×10⁻⁹ bar during the measurements. The wide scansurvey spectra and high energy resolution narrow scan spectra wererecorded at room temperature.

A 180° hemispherical energy analyzer model PHOIBOS-150 and a set of ninechannel electron multipliers MCD-9 was adopted for scanning. Theanalyzer was operated in Fixed Analyzer Transmission (FAT) and mediumarea lens modes at pass energy of 30 eV, step size of 1.0 eV for surveyscans. The pass energy was set at 20 eV with step size of 0.025 eV anddwell times of 0.1 sec for narrow or high resolution scans.

As the standard practice in XPS studies, the hydrocarbon C^(1s) line(284.6 eV) corresponding to C—C bond has been used as binding energyreference for charge correction. The high energy resolution spectra wereobtained under analysis conditions that would give a FWHM=0.85 eV fromthe Ag 3d_(5/2) signal of freshly argon ion etched silver (Ag) sample.

The surface wettability of the fibrous mat was analyzed according towater contact angle measurement performed by drop shape analyzer (DSA100). The BCCM was composed of 90% Dulbecco's Modified Eagle's Medium(DMEM) and 10% fetal bovine serum (“FBS”). A 2 ul of liquid was slowlydropped on the surface of the fiber mash then contact angle wasdetermined as an angle between the drop contour and the projection ofthe surface (baseline).

The protein attachment test was performed by the fibrous mats which werecleaned by DPBS (Dulbecco's phosphate-buffered saline). The fibrous matswere placed in 10% of FBS in DMEM for 24 hours. The samples were washedagain by DPBS to remove unattached proteins. The mats were placed afterwashing in 2% of SDS (sodium dodecyl sulfate) for 3 hours to release theattached proteins. The protein concentration was measured according tothe UV adsorption using the Nanodrop 2000. Also, the calculation wasbased on UV absorbance at 280 nm wavelength, where BSA (Bovine SerumAlbumin) was used as reference.

Biocompatibility test. The scaffolds collected on circular glass coverslips (diameter 12 mm) by two hours of electrospinning, were initiallysterilized under UV for 1 h followed by PBS washes (thrice for 5 mineach). The scaffolds were then washed with 100% ethanol for 10 min andagain washed thrice with phosphate buffered saline. The scaffolds werethen primed in the culture media and incubated at 37° C. incubator in a5% CO₂ in air atmosphere for 24 h to ensure no contamination. Thescaffold thickness was about 0.2 mm. A preferred range for scaffoldthickness is about 0.05 to 2 mm depending on the particular vasculartissue engineering application such as prosthesis of aorta, arteries andother smaller blood vessels.

Subsequently, the media was removed and fresh medium containing dermalfibroblasts (2×10⁴ cells/well) of a 24 well tissue culture plate wasadded to the four different scaffolds and cultured at standard cultureconditions of 37° C. in a 5% CO₂ incubator with regular changes of mediaevery 48 h.

After culturing for 7 days, the scaffolds were stained using celltracker fluorescent probe (CMFDA, c7025) to visualize the cellattachment on to the scaffolds. A fluorescent dye (5 μM) was added tocells in fresh media and incubated for 30 min under standard cultureconditions. The media containing the dye was then removed and thescaffolds containing the cells washed once with PBS to remove any tracesof the unbound dye and then the cell were fixed in 70% ice-cold ethanolfor 10 min. After phosphate buffered saline washes the cells wereincubated with 4′-6-Diamidino-2-phenylindole (DAPI) and greenflorescence protein (GFP) for 5 min at room temperature and washed withphosphate buffered saline again. The cells were then studied usingfluorescent microscope (EVOS® FL Imaging System, Thermo FisherScientific).

The cell proliferation assay was performed using an MTT reagent kit(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; Sigma).The 10 mL MTT reagent (final concentration of 0.5 mg/mL) was added tothe medium in the culture dishes and the dishes were incubated for 4 huntil a purple precipitate was visible. The medium was then removed and100 mL of the detergent reagent was added into the dishes and incubationcarried out in the dark for 2 h. The absorbance at 570nm wasspectrophotometrically measured using a microplate reader (SpectraMaxi3, San Jose, USA) with a reference wavelength of 570 nm.

FIG. 1 shows schematic representation of coaxial electrospinning setupfor the development of hollow fiber. The setup consisted of twoindependent syringe pumps which allowed the control of feed rate for thecore and shell solutions. The solutions were delivered through singlemetal needle tip with an 18 gauge outer needle and a 27 gauge innerneedle. Furthermore, the inner and outer solutions experience theelectrical field simultaneously during the electrospinning process. Theglycerol and mineral oil were used as core solution to serve astemplates and were removed after electrospinning the fibers.

The process parameters were selected and carried out by initiallysetting the shell feed rate at 0.5 mL/h and core feed rate at 0.1 mL/h.During the process the formation of the beads occurred in the resultantfibers. The formation of beads happened may be attributed to the shellsolution experiencing shear stress from both outer and inner wallresulting in insufficient feed rate for Tailor cone formation. Thereforethe feed rate of the shell solution was increased to 1 mL/h to maintainthe stable electrospinning process.

Chloroform and acetone (3:1) were selected as solvents for the solidfiber preparation. It was noticed that the needle was blocked by quicklydrying shell polymers in less than 15 seconds. The quick drying may beattributed to combined effects of needle configuration in coaxialelectrospinning and the solvent system.

Due to different capillary configuration in coaxial electrospinning, thesolvent evaporation was slowed down and acetone was replaced withethanol in the coaxial process to avoid unstable electrospinningconditions. In this stage, the inventors also tried to lower thevoltage. However this produced an adverse effect on the fibermorphology.

Subsequently, controlling the feed rate of the core solution was foundto be detrimental for successful preparation of hollow fiber usingcoaxial electrospinning. The initial experiments were carried out byvarying the core feed rate with increment of 0.1 from 0.1 mL/h to 0.3mL/h. Among the different core feed rate, 0.1 mL/h showed acceptableresult while higher feed rate caused formation of the beads. Theformation of beads implies that the feed rate needs to be controlledmore precisely. Based on this the inventors were able to determineadvantageous conditions for suitable development of hollow fibers.

The cross-sections of the hollow fibrous scaffolds prepared by usingglycerol as a core template are shown by FIG. 2 and demonstrate that0.05 mL/h of core feed rate is appropriate to develop hollow fiber butthe fiber walls were found to be thick.

Increasing the core feed to 0.08 mL/h resulted in larger opening of thehollow and relatively uniform fiber morphology. However, furtherincreasing of the feed rate beyond 0.1 mL/h led to the formation ofcollapsed fiber and fiber fusion.

This phenomenon may occur during the continuous fiber depositionprocess. During electrospinning the shell solution quickly dries duringdeposition. This leads to formation of complete solid phase in the shellof the fiber. However, the evaporation rate of glycerol was very low soit remained in liquid phase in the core throughout the electrospinningprocess. This might cause tearing of the shell layer and effluence ofglycerol to create fiber fusion Therefore, a high core feed rate ofglycerol can cause heavy fiber formation with easily deformable liquidphase fiber lumen. The initially deposited shell layer should bemechanically strong enough to bear upper pressure of the layers forcontinuous fiber deposition to avoid collapse.

A similar observation was made in the case of mineral oil using a corefeed rate of 0.1 mL/h (FIG. 3). The inventors believe that this may bedue to the low viscosity of the mineral oil compared to glycerol.Additionally, it was found that the core feed needed to be controlled ina narrower range in order to maintain good fiber morphology withoutcollapsing and fiber fusion. These results show that the feed rate ofglycerol at 0.08 mL/h and feed rate of mineral oil at 0.1 mL/h may beselected to prepare hollow composite scaffolds. FIG. 4 shows that theseconditions resulted in good hollow fiber morphology.

FIG. 5 shows the fiber size distribution of the hollow and solid fibrousscaffolds. The average fiber size in the scaffolds was found to be twicehigher in comparison to PLA/PBS (FIG. 5A) and PLA/PBS/CNF (FIG. 5B)scaffolds of solid fibrous. The difference in sizes may be due to thepresence of a core layer during the fiber flight time which creates aresistivity against fiber thinning by the electrical field.

Another potential reason is use of a different configuration of a Tailorcone during the coaxial electrospinning. In the solid fiber fabricationthe polymer droplet forms a completely polarized Tailor cone which hasinfluence on the size of the fiber. In case of coaxial electrospinningpolarization of the Tailor cone is disturbed by the core solution.Therefore the size of fiber became larger in this case. It should benoted that this phenomena cannot be applied for the coaxialelectrospinning in which both core and shell solutions involve spinning.The electric field influences both inner and outer solutions thatcontrol the overall fiber morphology and fiber size.

The average diameter of the cores in the hollow fibrous scaffolds wasmeasured using “Digimizer” software from 10 different places of SEMimage. Table 2 shows that both core templates had a similar hollow sizewhich was nearly 50% of average fiber size of the scaffolds. However,the core templates showed completely different results on the porositytheir fiber walls.

In the case of templates made using a glycerol core only few large porescould be observed and their dispersion was inconsistent as shown in FIG.6. On the other hand, mineral oil generated uniformly dispersed pores inthe fiber wall. This uniform dispersion may be due to the viscositydifferences between glycerol and mineral oil. It was noted that bothglycerol and mineral oil were slightly soluble with the shell solvent inthe presence of ethanol.

The high viscosity of the glycerol produces a stable one directionalflow in the electrospinning. However, the less viscous mineral oilgenerated spray and partly mixed with the ethanol. This spraying andpartial mixing encouraged more pore formation on the fiber wall.

Both blended and composite scaffolds were produced having 50 nm pores.The pores in case of blended scaffold are more uniform and spherical ascompared to the composite scaffolds. The geometry of the pores could beattributed to the presence of CNF in composite scaffold which preservesits shape and orientation.

TABLE 2 Average diameter of the cores in the hollow fibrous scaffoldsThe scaffold name Fiber core diameter GH 475 ± 52 nm (PLA/PBS) OH 485 ±66 nm (PLA/PBS) GC 327 ± 36 nm (PLA/PBS/CNF) OC 356 ± 47 nm(PLA/PBS/CNF)

FIG. 7 illustrates the XPS spectra of carbon and oxygen in the hollowfibrous scaffolds. The PLA and PBS were formed by the covalent bondingof carbon, hydrogen and oxygen. Among them, hydrogen cannot be detectedby XPS because it has no core electrons; see. Stojilovic, N., Why Can'tWe See Hydrogen in X-ray Photoelectron Spectroscopy? Journal of ChemicalEducation, 2012. 89(10): p. 1331-1332, incorporated herein by referencein its entirety.

Except for hydrogen, each PLA monomer consisted of two C—C—C bonds, oneC—C—O bond, one O═C—O bond, one O═C bond and one C—O—C bond. Similarbonds exist in each PBS monomer with double the ratio of the PLAmonomer.

The PLA/PBS were homogeneously blended at a 50/50 concentrationresulting in a required ideal ratio of 33.33% C—C—C bonds and 16.67%other carbon and oxygen bonds, as shown in Table 2.

From the XPS spectra, two main peaks were observed: the binding energyof the first peak was at 284.79 eV which corresponds to a carbon atom,and the binding energy of another peak was at 532.64 eV which relates toan oxygen atom; see Chastain, J., R. C. King, and J. Moulder, Handbookof X-ray photoelectron spectroscopy: a reference book of standardspectra for identification and interpretation of XPS data. 1995:Physical Electronics Eden Prairie, Minn., incorporated herein byreference in its entirety.

As described above in the XPS results, when the PLA/PBS arehomogeneously blended with 50/50 concentration, this results in arequired ideal ratio of 33.33% C—C—C bonds and 16.67% of other chemicalbonds (CCO, COO, OH/COC, —OC). In case of CNF incorporation OHconcentration is supposed to be higher due to dominance of OH in thePLA/PBS/CNF composite. The chemical bond composition of each atom wascalculated and the results are presented in the Table 2. The bondcomposition of the scaffold is similar to the ideal bond composition.For example, percentage of C—C—C bond is approximately 35% which is veryclose to ideal percentage 33.33%. For the PLA/PBS scaffold percentage ofother bonds are near to 16.67%. While 25% of OH bond is observed for thePLA/PBS/CNF composite scaffold which highlights dominance of OH in theCNF. Therefore the result implies coherency of the individual materialin the composite nanofiber.

TABLE 2 Chemical bond composition and Binding Energy extracted from XPSpeaks Binding Concentration (at. %) Name energy PLA/PBS PLA/PBS/CNF O 1s532.64 35.182 40.323 C 1s 284.79 64.818 59.677 O ls_1_O═C 532.08 18.43315.032 O 1s_2_C—O—C/OH 533.48 16.749 25.291 C 1s_1_C—C—C 284.8 35.41435.192 C 1s_2_C—C—O 286.59 15.396 12.824 C 1s_3_O—C═O 288.82 14.00811.662

When compared to PLA/PBS blend scaffolds to the PLA/PBS/CNF compositescaffolds containing CNF exhibited higher oxygen concentration.

In case of a PLA/PBS blend, the XPS characteristic of the hollow fibrousscaffolds was found to be similar to that of solid fibrous scaffold.When compared to PLA/PBS blend scaffolds, PLA/PBS/CNF compositescaffolds containing CNF exhibited higher oxygen concentration.

Particularly, the intensity of 0-H/O-C bond for the composite scaffoldssignificantly increased when compared to PLA/PBS scaffolds. The increasein intensity could be due to dominance of OH bonds in the CNF; seeKrouit, M., J. Bras, and M. N. Belgacem, Cellulose surface grafting withpolycaprolactone by heterogeneous click-chemistry. European PolymerJournal, 2008. 44(12): p. 4074-4081, incorporated herein by reference inits entirety.

This result confirms that the materials were homogenously dispersed inthe hollow fibrous scaffolds. Whereas overall intensity of the XPSspectra in case of oil templated hollow fibrous scaffolds is lower thanthe glycerol templated hollow fibrous scaffolds. The lower intensitycould be result of high porosity of the fiber wall when oil is used astemplate.

The developed hollow fibrous structure improves the wettability of thescaffold as shown by FIG. 8. For the PLA/PBS blend scaffolds, the watercontact angle (WCA) was decreased by ˜30° in case of GH and by ˜40° incase of OH when compared to solid scaffold.

Similar trends were observed for the composite scaffolds. The WCA of41.6±2.1° is found in OH scaffold which is the lowest among the hollowfibrous scaffolds. This could be because of uniformly dispersed pores onthe fiber wall. This result is favorable because the uniformity in thestructure of pores is beneficial for better dispersion of liquids in ascaffold; see Francis, L., et al., PVDF hollow fiber and nanofibermembranes for fresh water reclamation using membrane distillation.Journal of Materials Science, 2014. 49(5): p. 2045-2053, incorporatedherein by reference in its entirety.

The hydrophilicity of the hollow fibrous scaffolds was found to be muchhigher than that of solid fibrous scaffolds within 40˜80° of WCA; seeArima, Y. et al., Effect of wettability and surface functional groups onprotein adsorption and cell adhesion using well-defined mixedself-assembled monolayers. Biomaterials, 2007. 28(20): p. 3074-3082; andWei, J., et al., Influence of surface wettability on competitive proteinadsorption and initial attachment of osteoblasts. Biomedical Materials,2009. 4(4): p. 045002, each incorporated herein by reference in theirentirety. The improved mobility of liquids enables the interaction ofthe cells with the scaffolds and enhances the cell adhesion andproliferations.

Moreover as shown by FIG. 9, hollow fibrous structures have an impact onprotein adsorption capacity of the scaffolds. The mineral oil templatedhollow fibrous scaffolds showed double protein adsorption capacity whencompared to solid fibrous scaffolds. The enhanced adsorption capacitymay be due to the increased surface area because of hollow conduits andmicropore formation on the fiber wall. Also, higher hydrophilicityincreases the protein adsorption capacity of the scaffolds.

FIG. 10 depicts GFP/DAPI staining images of the hollow fibrous scaffoldsafter 7 days. All scaffolds supported better adhesion and dispersion ofthe cells through entire area. The mineral oil templated hollowscaffolds showed more occupied cell attachment compared to the glyceroltemplated hollow scaffolds.

In the case of GH and GC scaffolds, produced using a glycerol core, anumber of black spots can be observed from the staining image whichimplies that no cell attachment occurred in those areas.

Meanwhile, OH and OC scaffolds, produced using a mineral oil core,showed completely overlapped cell attachment without any black spots.This could be strongly correlated with overlarge surface area of themicroporous hollow fibrous scaffolds. The enhanced surface energy andenriched protein adsorption capacity of the scaffold are thecontributing factors for the better cell adhesion performance of OH andOC scaffolds; Arima et al., Electrospun 1,6-diisocyanatohexane-extendedpoly(1,4-butylene succinate) fiber mats and their potential for use asbone scaffolds. Polymer, 2009. 50(6): p. 1548-1558; Garcia-Orue, I., etal., Chapter 2-Nanotechnology approaches for skin wound regenerationusing drug-delivery systems A2-Grumezescu, Alexandru Mihai, inNanobiomaterials in Soft Tissue Engineering. 2016, William AndrewPublishing. p. 31-55; and Tallawi, M., et al. Poly (glycerol sebacate)Poly (butylene succinate-dilinoleate) Blends as Candidate Materials forCardiac Tissue Engineering. in Macromolecular Symposia. 2013. WileyOnline Library, each incorporated herein by reference in their entirety.

The cell proliferation result for the hollow fibrous scaffolds at 7 and14 days by MTT assay can be observed in FIG. 11A. Similarly, in thesolid fibrous scaffolds the rapid increase in cell proliferation wasachieved in the first week and followed by a slower increase in thesecond week. Whereas, the hollow fibrous scaffolds showed a higher cellproliferation rate compared to the solid fibrous scaffolds (FIG. 11B).The highest cell proliferation rate is found in case of OH scaffoldwhich is even higher than that of OC scaffolds. The improvedproliferation rate shows the importance of uniform pore formation on thefiber wall. The microporous hollow fibrous scaffolds can provide forbetter transport of nutrients, oxygen and other important biomoleculesto the cells. The cells with the help of newly developed scaffold can beprovided with an advantageous microenvironment for the growth,propagation and viability.

As demonstrated herein, homogenously blended microporous hollow fibrousscaffolds were successfully synthesized from PLA/PBS blends and CNFreinforced composites using mineral oil as a core template. Among thecoaxial electrospinning parameters core and shell feed rate were foundto be the most determining parameters to generate uniform hollow fiberwithout fusion and beads. The mineral oil and glycerol both as coretemplates allowed hollow fiber formation form PLA/PBS blends and CNFreinforced composites in electrospinning. The mineral oil resulted information of uniformly dispersed microspores on the fiber wall. Also,the surface composition of the hollow fibrous scaffolds was notinfluenced by the core templates. The high porosity in the fiber wallwas confirmed for the oil templated hollow fibrous scaffolds. Thesurface properties such as wettability and protein adsorption capacitywere remarkably improved in the presence of microporous and hollowfibrous structure. Finally, the microporous hollow fibrous scaffoldsshowed enhanced cell adhesion and proliferation performance than thesolid fibrous scaffolds.

1-10. (canceled)
 11. The method of claim 12, wherein the hollow fiberscaffold is a portion of a sterile or aseptic patch, packing, dressingor bandage applied to the tissue in need of repair, and wherein thehollow fiber scaffold comprises cells or one or more biologically activeagents.
 12. A method for repairing living tissue, comprising applying ahollow fiber scaffold on, in or around living tissue in need of repair,wherein the hollow fibers of the hollow fiber scaffold fibers have ahollow core and a peripheral shell, wherein the peripheral shellcomprises a mixture of homogenously dispersed polylactic acid (PLA),polybutylene succinate (PBS) and cellulose nanofibers (CNF); wherein thehollow fibers have an average diameter ranging from 400 to 1,000 nm andthe hollow core has an average diameter ranging from 300 to 900 nm. 13.The method of claim 12, wherein the living tissue in need of repair is avascular tissue.
 14. The method of claim 12, wherein the living tissuein need of repair is an epithelial tissue. 15-20. (canceled)
 21. Themethod of claim 12, wherein the peripheral shell has pores ranging insize from 25 to 100 nm in diameter.
 22. The method of claim 12, whereinthe peripheral shell has pores ranging in size from 40 to 60 nm indiameter.
 23. The method of claim 12, wherein the peripheral shell hasuniformly distributed pores wherein an average size or numbers of poresin different areas of the peripheral shell varies by no more than 10%.24. The method of claim 12, wherein the hollow fiber scaffold has aninterconnected macroporous structure.
 25. The method of claim 12,wherein the hollow core and micropores of the peripheral shell provideenhanced diffusion of nutrients and oxygen to the cells grown on or withthe scaffold as compared to cells grown on scaffolds comprising solidfibers.